Saposhnikovia divaricata is a traditional Chinese medicine in China, which is widely used in clinic. The root of S. divaricata is often used as medicine, but little research has been done on its other tissues.In this study, the contents of root and leaf of S. divaricata were determined by HPLC, the differentially expressed genes were screened by transcriptome sequencing at molecular level, and then verified by network pharmacology.The results showed that the content of 4’-O-β-D-glucosyl-5-O-methylvisamminol in the leaves was significantly higher than that in the roots, which was about 3 times higher than that in the roots. In addition, 10 differentially expressed key enzyme genes were screened in plant hormone signal transduction, phenylpropanoid and flavonoid biosynthetic pathways. C4H and CYP98A were up-regulated in root, while F3H was down-regulated in root. They can be used as important candidate genes for the mechanism of quality difference of S. divaricata. Finally, network pharmacological validation showed that 5-O-methylvesamitol plays an important role in the treatment of ulcerative colitis.S. divaricata.These findings not only provide insight into flavonoid biosynthesis in S. divaricata associated molecular regulation, but also provide a theoretical basis for the development and utilization of  With the depletion of resources and the deterioration of ecology, the study on the comprehensive utilization of non-traditional medicinal parts of common medicinal materials has attracted more and more attention. For example, ginseng is commonly used for its roots but studies have shown that the stems and leaves of ginseng also have good antioxidant activity . It is oSaposhnikovia divaricata (S. divaricata) is derived from the dried roots of Saposhnikovia divaricata (Turcz.) Schischk and used as a Chinese herbal medicine for treating respiratory, immune, and nervous system diseases , and then transferred to flavonoid biosynthetic pathway by Chalcone synthase(CHS), and Chromone was produced by a series of reactions  at the end of August 2020. The collected samples were divided into two tissue sites, root and leaf, as shown in Samples were collected from three-year-old 2.2O-glucosylcimifugin (80681-45-4), cimifugin(37921-38-3), 4’-O-β-D-glucosyl-5-O-methylvisamminol(84272-85-5), and sec-O-glucosylhamaudol(80681-44-3) were all supplied by Shanghai YUANYE Biotechnology Co., Ltd. The purity of each ingredient was greater than 98% as determined by HPLC. Appropriate amounts of prim-O-glucosylcimifugin, cimifugin, 4’-O-β-D-glucosyl-5-O-methylvisamminol, and sec-O-glucosylhamaudol were precisely weighed, and then methanol was added. High-performance liquid chromatography was performed using an Agilent 1,200 series high-performance liquid chromatography system , equipped with an autosampler and a UV detector with a C18 column . Gradient elution was performed using solvent A (100% methanol) and solvent B (100% water) at 30°C, according to the following gradient program: 0–20 min, 30% A; 20–25 min, 45% A; 25–50 min, 60% A; 50–55 min, 90% A; and 55–60 min, 100% A. The flow rate was maintained at 1.0 ml/min, the sample injection volume was 10 μl, and UV absorption was measured at 254 nm. A mixed solution containing all reference substances was prepared and diluted with methanol to obtain 4 reference substance solutions of different concentrations. Mixed reference solutions of different concentrations were used to draw the standard curve. As shown in 2 > 0.997) and a good linearity over a wide range. The HPLC chromatogram of the S. divaricata reference solution is shown in After drying the samples, these were ground into a powder. The ground powder was filtered through a 60-mesh sieve and then weighed in three replicates, each weighing 1.0 g. Chromogen ketones were extracted ultrasonically , extracted three times with methanol, filtered in a funnel, and then the filtered liquids were combined. After the filtrate was evaporated in an evaporating dish and transferred to a 5 ml volumetric flask, the volume was adjusted to 5 ml and the solution was filtered using a 0.22 μm filter. prim-2323.1Total RNA was extracted using Trizol reagent  following the manufacturer’s procedure. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 1000 Nano LabChip Kit  with RIN number >7.0. Poly (A) RNA is purified from total RNA (5ug) using poly-T oligo-attached magnetic beads using two rounds of purification. Following purification, the mRNA is fragmented into smallpieces using divalent cations under elevated temperature. Then the cleaved RNA fragments werereverse-transcribed to create the final cDNA library in accordance with the protocol for the mRNASeq sample preparation kit , the average insert size for the paired-endlibraries was 300 bp ( ± 50 bp). And then we performed the paired-end sequencing on an Illumina Novaseq™ 6000 at the  following the vendor’s recommended protocol.2.3.2http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). including the Q20, Q30 and GC-content of the clean data. All downstream analyses were based on clean data of high quality. De novo assembly of the transcriptome was performed with Trinity 2.4.0 ,Gene ontology (GO)(http://www.geneontology.org), SwissProt(http://www.expasy.ch/sprot/), Kyoto Encyclopedia of Genes and Genomes (KEGG)(http://www.genome.jp/kegg/) and eggNOG(http://eggnogdb.embl.de/) databases using DIAMOND protein database  was used as a housekeeping gene to estimate the relative gene expression using the 2−ΔΔCt method. The primer sequences used in this study are shown in The instructions of the TaKaRa MiniBEST Universal RNA Extraction Kit were followed for total RNA extraction , and the PrimeScriptTM RT Master Mix Kit (TaKaRa) was used for reverse transcription. RT-qPCR was performed on a 96-well plate using an Agilent Technologies Stratagene Mx3000P thermal cycler and a SYBR Green-based PCR kit. Each reaction involved 1 μl of cDNA template (1 mg/ml), 10 μl of SYBR Green Mix (TaKaRa), 1 μl of forward primer (1 mM), 1 μl of reverse primer (1 mM), and 7 μl of ddH2O. The thermal conditions were as follows: 95°C for 3 min, followed by 40 cycles at 95°C for 5 s, 55°C for 32 s, and 72°C for 20 s. The melting curve was obtained by gradually increasing the temperature from 55°C to 95°C at a heating rate of 0.1°C/s. RT-qPCR analysis was performed using three biological replicates. To validate the reliability of transcriptome data, 10 differentially expressed genes with high or low expression levels were randomly selected for RT-qPCR validation. 242.4.1S. divaricata is taken as an example. The TCMSP (https://tcmspw.com/) database was used to collect the chemical composition of S. divaricata, and the Gene Cards (https://www.Genecards.org/) database was used to collect ulcerative colitis genes, the Uniport (http://www.uniprot.org/) database was used to standardize proteins.In the section on network pharmacology, the treatment of ulcerative colitis with 2.4.2S. divaricata were collected through the TCMSP database, and the two indicators of oral bioavailability (OB) ≥ 30% and drug-like index (DL) ≥ 0.18 were used as screening thresholds. Finally, we combined the literature to determine the final drug active ingredient. The potential target proteins corresponding to the active components were collected one by one, and the potential targets corresponding to the drugs were obtained. “Ulcerative colitis” was used to search for ulcerative colitis genes in the Gene Cards database. After processing and de-weighting the collected disease targets, the ulcerative colitis targets will be obtained.All the active ingredients of 2.4.3S. divaricata. Cytoscape 3.8.0 software was used to construct the visualized network of “Drug-active component-target-disease”.The drug-target and disease-target were mapped by R language related Venny package, and the Venny diagram was drawn to get the ulcerative colitis targets of 33.1S. divaricata. The content of prim-O-glucosylcimifugin in the root of S. divaricata was significantly higher than that in the leaves, about 2.5 times as much as that in the leaves. However, the content of 4’-O-β-D-glucosyl-5-O-methylvisamminol in leaves was significantly higher than that in roots, about 3 times as much as that in roots. There was no significant difference in the content of cimifugin between the two groups. Sec-O-glucosylhamaudol was found in the roots, but not detected in the leaves. Based on these results, we found that the contents of chromogenin in the roots and leaves of S. divaricata were significantly different and further speculated that there must be a relationship between the transformation and accumulation of effective components. Therefore, transcriptome sequencing was used to further explore the molecular mechanism of their differences.As shown in 3.23.2.1S. divaricata were sequenced by Illumina NovaseqTM 6000 platform. The results were as shown in Six cDNA libraries of root and leaf samples of S. divaricata was not well aligned, the comparative analysis of the non-reference transcriptome was used in this study. Trinity software was used to de novo assemble all clean reads from the six libraries into contigs and reflect reads back to contigs, removing redundancy, then defining the longest transcript as Unigene. Finally, 48219 Unigenes were obtained, and the N50 length was 1585 nt. The sequencing and assembly results are shown in Because the reference genome of 3.2.2In order to understand the function information of the assembled Unigenes, Diamond Software was used to perform function annotation. The database of comparison included NR, GO, Kegg, Pfam, Swiss-Prot, Egg Nog. The result of function annotation was shown in S. divaricata have the highest homology with Daucus carota.The NR annotation results are shown in Go Function annotation classification information is shown in The results of KEGG functional annotation to metabolic pathways are shown in The results of the eggNOG functional annotation classification are shown in 3.2.3A total of 5447 DEGs were screened in the root vs. leaf comparison group, of which 2029 DEGs were upregulated and 3418 DEGs were downregulated. The number of down-regulated genes was higher than that of up-regulated genes in the root vs. leaf comparison group, which indicated that the differential genes were low-expressed in root and high-expressed in leaf. ≥30% and drug-like activity (DL)≥0.18. Prim-O-glucosylcimifugin, 4’-O-β-D-glucosyl-5-O-methylvisamminol, cimifugin, and sec-O-glucosylhamaudol are widely considered as active compounds in S. divaricata due to their high content and strong pharmacological activities. Although their oral bioavailability is less than 30%, they are considered as bioactive compounds and can be used for further analysis  can be found below: YC, TZ and CC conceived and designed the experiments. YC and TZ performed most of the experiments and completed the first draft. YC, TZ, CC, ZX and CL conducted the experiments and carried out the analysis. All authors contributed to the article and approved the submitted version.


Therefore, both star-shaped PCL-PEGs were investigated as cargo device for ciprofloxacin. No significant differences were observed in terms of drug entrapment efficiency (>95%) and drug release, characterized by a pronounced burst followed by a slow sustained release, only slightly affected by the co-polymer architecture. This result was also confirmed with curve fitting via the Korsmeyer–Peppas model. Lastly, good antibacterial properties and biocompatibility exhibited in both star-shaped PCL–PEG co-polymers suggest a promising use for oral delivery applications.The administration of poorly water-soluble drugs represents a relevant problem due to the low body fluids transport efficiency through hydrophilic hydrogels. Star-shaped co-polymers, i.e., amphiphilic polymers such as those with a hydrophobic core and a hydrophilic outer shell, can be used to improve weak interactions with drugs, with relevant benefits in terms of administration and controlled delivery. In this work, two different co-polymers, four-arm star-shaped PCL–PEG and six-arm star-shaped PCL–PEG, were synthesized via ring-opening polymerization to be loaded with ciprofloxacin.  The development of effective medicinal products from poorly water-soluble drugs continues to be a challenging task with the current pharmaceutical technology; however, this is crucial for the treatment of a wide range of diseases. Approximately 40% of drugs currently approved for market use and almost 90% of molecules still in the discovery phase suffer from poor water solubility, which ultimately results in limited absorption and low bioavailability when orally administered ,4,5,6,7.In this context, polyesters/polyether-based amphiphilic star-shaped polymers, such as poly-caprolactone-b-poly(ethylene glycol) (PCL–PEG), enable the increase in the encapsulation of a hydrophobic drug via a hydrophobic–hydrophobic interaction or the conjugation of a drug with PCL arms ,19,20. TIn this study, amphiphilic star shapes of polycaprolactone–polyethylene glycols consisting of four and six arms were synthesized. In detail, the hydrophobic, slowly degradable arms of PCL were grafted to hydrophilic PEG segments to form a star-shaped architecture. Ciprofloxacin (Cipro) was used as a model poorly water-soluble drug to validate the use of star-shaped PCL–PEG as a carrier for the oral administration of hydrophobic drugs. P. aeruginosa (ATCC 27853), E. coli (ATCC25922), E. faecalis (ATCC29212), and S. pyogenes (ATCC19615) were procured from the American Type Culture Collection (ATCC), Manassas, VA, USA.ε-Caprolactone, succinic anhydride, pentaerythritol, di-pentaerythritol, 4-(dimethylamino)pyridine (DMAP), 1,3-dicyclohexylcarbodiimide (DCC), ciprofloxacin 99%, and carbomer 940 were purchased from Across Organic  and were used without further purification. Monomethoxy poly(ethylene glycol), (mPEG) with Mn = 5.0 kDa, and Tin(2-ethylhexanoate)2 (Sn(Oct)2) were purchased from Sigma-Aldrich . Methyl paraben, ethyl paraben, and trifluoroethanol (TFE) were purchased from Merck. Pvt. Ltd. , and trieathanolamine (TEA) was purchased from Q-rec . All chemicals used in this study were of analytical grade and used without further purification. Bacterial strains of In the preparation of star polymer, two types of initiators were used. Pentaerythrytol was used to develop four-arm star polymer, and di-pentaerythritol for six-arm star polymer . The molFor the four-arm star-shaped PCL (4Star-PCL) and six-arm star-shaped PCL (6Star-PCL), approximately 10 g (0.087 mol) of ɛ-caprolactone was mixed with the pentaerythritol/di-pentaerythritol (0.27 mmol) in a 100 mL round bottom flask. Then, the mixture was heated to 110 °C, and upon homogeneity, 0.005 g (0.1 mmol) stannous octoate catalyst was added. The flask was purged with nitrogen and covered with parafilm. All reactions were maintained using a silicone oil bath at the specified reaction temperature of 110 °C for approximately 24 h. The polymers were isolated via precipitation into cold diethyl ether, followed by several washes with copious amounts of the precipitating solvent, and dried under vacuum for 48 h. PCL-PEG star polymer was then synthesized by reacting carboxylated mPEG, 4Star-PCL, and 6Star-PCL . mPEG-CO−1. 1H-NMR spectra of the polymers dissolved in deuterated chloroform (CDCl3) were recorded using a Varian 400 spectrometer at 400 MHz. In this case, Tetramethylsilane (TMS) was used as the internal reference, while all chemical shifts were reported in parts per million (ppm). Thermogravimetric TGA/DTG analysis was carried out using a Thermogravimetric Analyzer  on a crucible of aluminum containing 5 mg of sample. All the tests were performed by imposing a temperature range from 30 to 600 °C with a heating rate of 10 °C under nitrogen (N2) flow (50 mL/min). The differential scanning calorimetry (DSC) analysis was carried out using a Differential Scanning Calorimeter  under nitrogen flow (10 mL/min) by imposing a heating rate of 10 °C/min into a range from 30 to 400 °C. The glass transition temperature (Tg) and crystalline melting temperature (Tm) were recorded.The functional groups of star polymers were identified using Perkin-Elmer Spectrum 400FTIR/FT-NIR spectrometer. Solid samples were ground with anhydrous potassium bromide (KBr). Infrared spectra were recorded in the region 4000–400 cm4Star PCL-PEG and 6Star PCL-PEG (ca. 300 mg) were first diluted in TFE and then mixed with ciprofloxacin (90 mg) via magnetic stirring for 12 h until it formed a homogeneous mixture. Methyl paraben (15 mg) and propyl paraben (9 mg) were added in the formulation as preservative agent. Meanwhile, Carbopol resin was dispersed in 15 mL of deionized water for 12 h until it became a clear solution. Both mixtures were then mixed together and stirred for 24 h. The final mixture was neutralized with dropwise addition of trimethylamine until it formed a stable gel stabilized via storage for 24 h at room temperature. Standard formulation without ciprofloxacin loading was also prepared as control (STN6 and STN6).For all the formulations, pH was measured using digital pH meter . The equipment was calibrated via a standard buffer solution at 4.0, 7.0, and 9.0. The pH probe was dipped into the solution for two minutes until a stable pH reading was obtained. pH measurements were performed in triplicate, and the average value was considered. A Brookfield digital viscometer  was used to measure the gel viscosity. After 30 minutes of rest, a spindle rotation (level 4) of 50 rpm was applied to the solution, and the viscosity measure was collected after 2 min.2 = 0.9979) was reported in The prepared gels (100 mg) were dissolved in 100 mL of phosphate-buffered solution. The flask containing gel solution was shaken for 30 min on mechanical shaker. Then, the sample was analyzed via UV spectrophotometer. The calibration curve , i.e., zero-order model, will be considered for in vitro studies.The release kinetics of ciprofloxacin was investigated using four different models: zero-order model, first-order model, Hixson–Crowell, Higuchi, and Korsmeyer–Peppas model . The modt = k0 tZero-order release: Qt is the total drug release at time, t (in percentage concentration), and k0 is the constant of zero-order model (in concentration/time).where Q0 − kt/2.303First-order release model: Log C = Log C0 is the initial concentration of the drug, k is the constant of first-order model, and t is the time.where Qt = KH t1/2Higuchi model: Qwhere Q is the amount of drug released in time, and t and KH are the Higuchi dissolution constants. The amount of drugs released reported as function of the square root of time, fitted a straight line.t/M = kkp tnKorsmeyer–Peppas release model: Mt and M are the amount of drug at time t and loaded into the drug delivery system, respectively, and k is the kinetic constant related to the drug delivery system.where M“n” is the release exponent that depends on the type of transport, geometry, and polydispersity of the solute and is strictly related to the solute transport mechanism: Fickian diffusion (n = 0.5), anomalous transport (0.5 < n < 1), and pseudo-Fickian diffusion (n < 0.5).The bacteria were cultured on Mueller Hinton Agar (MHA) for 24 h at 37.5 °C. In vitro antibacterial activity in the gels was evaluated using agar well diffusion method . A steri® CRL-2014TM—American Type Culture Collection—Manassaa, VA, USA), were used for biocompatibility studies. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum.Human gingival fibroblasts, HGF-1 (ATCC4 cells/mL) was seeded into 96-well plates and incubated for 4 days until it reached the confluence. Then, culture medium was removed, washed with phosphate buffer, and replaced with complete (100 µL) medium containing 5, 8, and 10 µg/mL of gel solution for all four investigated types. Experiments were conducted in triplicate (n = 9).4SF, 6SF, STN4, and STN6 samples were dissolved in 10 mL of DMEM medium (10 µg/mL). Then, the medium was filtered through a 0.22 mM filter paper and kept as a mother solution. Two dilutions were prepared to reach a final concentration of samples equal to 8 µg/mL and 5 µg/mL. HFG-1 . The results were presented as mean ± standard deviation.The cytotoxicity tests were performed using Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The MTT solution, 10 µg/mL per well, was kept in incubator at 37 °C, 5% COt-test was used for the statistical analysis among the groups. Statistical significance was considered at p < 0.05.All data were expressed as average ± standard deviation. Data analysis was performed using Origin Pro 8.5.0. The Student’s −1. In both cases, the C-O stretched bands appeared around 1172 cm−1. The C-H band of the methyl group of the PCL was detected from 2943 to 2865 cm−1 in the case of 4Star PCL and from 2944 cm−1 to 2865 cm−1 in the case of 6Star PCL. The presence of a PCL backbone band confirmed the ROP of cyclic ɛ-caprolactone. The presence of a weak hydroxyl band at ~3500 cm−1 is probably due to the low content of the -OH in the compounds  are referred to PCL backbone, confirming ROP of ɛ-caprolactone monomer. The polymerization of each arm from the core is assumed to be the same since the active group of each arm is the hydroxyl group that initiates the ROP of caprolactone  denotes that the PCL backbone is attached to the central core. A triplet at 2.65 ppm  denotes the methylene group linker. This peak is slightly shifted to a downfield due to the de-shielding effect after PCL backbone attachment. A singlet at 3.4 ppm  is associated with the methoxy group for the end terminal in mPEG, while chemical shifts from 3.5 to 3.7 ppm  are referred to as poly (ethylene glycol) backbone. Meanwhile, a triplet at 4.0 ppm  is associated with the methylene ester on the PCL backbone [backbone . The 1H The thermal properties of the synthesized four- and six-arm star-shaped block co-polymer were examined using thermogravimetric analysis (TGA/DTG) and differential scanning calorimetry (DSC). The initial decomposition for both 4Star PCL and 6Star PCL is about 310 °C .3), leading to higher thermal stability compared to star PCL homopolymers due to hydroxyl terminal end groups (-OH), which are prone to rapid thermal degradation. The similar degradation rates in both star PCL homopolymers and star PCL-PEG are due to the same molar ratio of both PCL and PEG, even though they have different architectures. The results showed that the polymer architecture did not significantly affect the thermal degradation of the polymers. The observed differences in thermal stabilities of the synthesized polymers are consistent with their structures.Even though both PCL star homopolymers are approximately the same in terms of molecular weight, the length for each six- and four-arm star-shaped polymer is different, depending on its degree of polymerization. The star polymers PCL-PEG showed high thermal stability based on an initial decomposition temperature of 340 °C. The addition of mPEG-COOH to both star PCL increased the thermal stability of the polymer compared to the homopolymer itself. These thermal changes were expected since the addition of PCL increased the decomposition temperature of the co-polymers due to the high thermal stability of PEG . Star coThermal analysis using DSC was used to gain insight into the self-organization and the crystallization behavior of the star-shaped co-polymers in bulk. The melt crystallization of these co-polymers is rather complex since both PCL and PEG have close melting temperatures that lead to the occurrence of coincident crystallization of both components, or the crystallization of one block may affect the crystallization of the second block ,39,40. Am values of these co-polymers are very close to each other (59–60 °C), thus showing that the architecture does not significantly affect the thermal behavior of PCL-PEG star-shaped block co-polymers. Based on the DSC value, both block co-polymers gave slightly lower Tm compared to mPEG-COOH. Such a finding may be related to the higher molecular weight of PCL blocks, which crystallized first and consequently disturbed the organization of the PEG segment that altered the Tm value of PEG. This also shows that the Tm of PCL also were not affected by the increased PEG content since 6Star PCL-PEG has a higher number of PEG block compared to 4Star PCL-PEG. The Tm value of PCL in block co-polymers was almost identical to its star-shaped homopolymers, suggesting that PCL crystallization is barely affected by the presence of the PEG segment. Since there was no significant observable mPEG melting peaks in the co-polymer samples, it can be concluded that both star block co-polymers consisted of a semi-crystalline PCL phase, while PEG dispersed within the part of the semi-crystalline PCL phase [Both 4Star PCL and 6Star PCL were observed to possess crystalline melting points within the melting range of PCL . These bCL phase .Two formulations were prepared by loading ciprofloxacin in 4Star PCL-PEG (4SF) and 6Star PCL-PEG (6SF), respectively. The main challenge in preparing hydrogel formulation containing PCL is the hydrophobicity of PCL, and the co-polymerization of PCL with PEG is to increase the solubility of the star polymer in water to be incorporated into the formulation. TFE was chosen as the solvent to dissolve the star polymers and ciprofloxacin before mixing it with a water base carbomer. The ratio of the ciprofloxacin and TFE used is very crucial to make sure that the hydrogel formulation is in a homogenous form. After the addition of water to the mixture of star polymer, drugs, and carbomer, an opaque hydrogel formed with a smooth and homogenous appearance without any agglomeration. However, formulations that contain star-shaped homopolymer PCL, either four or six arms, did not form a homogeneous formulation as the polymer tends to agglomerate in the hydrogel. The introduction of hydrophilic polyether blocks, PEG, into PCL chains is a means to enhance the hydrophilicity of the star polymers compared with the homopolymer to form a homogenous mixture in the formulation. The hydrophobic blocks associate to form the inner region, whereas the position of the hydrophilic segments between the inner segment and the external aqueous medium led to an increase in hydrophilicity. Hence, the hydrophobic segment is stabilized via the hydrophilic outer segment, which serves as an interface between the bulk aqueous phase and the hydrophobic domain . The amoAll hydrogels have shown high drug entrapment efficiency with more than 98% encapsulation , indicatThe release of drugs is typically influenced by initial diffusion, followed by a combination of diffusion and degradation mechanisms . Both stEven though the 4SF and 6SF samples have similar release behaviors, the 6SF showed a slightly higher amount of drug released compared to the 4SF. This behavior may be attributed to the 6SF architecture, which has a relatively longer PCL block length and a higher number of PEG compared to 4SF. Mishra et al.  suggeste2, from The drug-release data of the formulations were examined for the applicability of various mathematical kinetic models to consider the suitable dissolution profiles of the samples ,47. The The “n” value  for the 2 values obtained from the mathematical modeling are not significantly closer to 1, the data serve as a preliminary study to obtain novel insight into the release kinetics of the drug model from the formulations. This data can be used for the further optimization of the formulation, especially the drug-loading and drug-release capacity, to ensure that it can be used effectively toward targeted application.Fick diffusion suggested that the hydrogel network was relaxed after the dissolution medium penetrated the hydrogel matrix. Then, the drug model was carried out using the medium that entered the matrix through the pores of the hydrogel matrix via diffusion ,51. ThisE. coli, P. aeruginosa, E. faecalis, and S. pyogenes) was performed via the zone of inhibition testing. All formulations containing ciprofloxacin showed activity against the selected Gram-positive and Gram-negative bacteria , in six-arm star polymers hydrogels slightly higher than the four-arms star polymer ones. Sustained drug release, reported for star polymer-based hydrogels, is well fit via the kinetics model following the Korsmeyer–Peppas release profile. An antibacterial study of the hydrogels confirmed good antimicrobial activity due to the action of ciprofloxacin and its sustained release from the hydrogels. The cytotoxicity tests showed a low cytotoxic response of the hydrogels suitable for biomedical applications. Hence, multiple arm star polymers can be considered promising for the development of novel carriers for oral delivery applications.In summary, the amphiphilic star polymers of four- and six-arm PCL-PEG with different architectures were successfully synthesized and fully characterized via


Patients with significant residual MR presented with a higher rate of all-cause mortality at 5 years compared with trivial MR . MR progression occurred earlier in the RA-r group, as 20 patients in the RA-r group vs. 6 in SV-r + RA-r group had a significant MR 2 years after surgery (p = 0.002). Conclusions. RA-r remains a surgical mitral repair technique with an increased risk of failure and mortality at 5 years compared with SV-r. The rates of recurrent MR are higher, and recurrence occurs earlier, with RA-r alone compared to SV-r. The addition of the subvalvular repair increases the durability of the repair, thus extending all of the benefits of preventing MR recurrence.Objectives. The current guidelines still do not include specific recommendations on the use of subvalvular repair (SV-r) for treatment of ischemic mitral regurgitation (IMR). Therefore, the objective of our study was to evaluate the clinical impact of mitral regurgitation (MR) recurrence and ventricular remodeling on long-term outcomes after SV-r combined with restrictive annuloplasty (RA-r). Methods. We performed a subanalysis of the papillary muscle approximation trial, studying 96 patients with severe IMR and coronary artery disease undergoing restrictive annuloplasty alongside subvalvular repair (SV-r + RA-r group) or restrictive annuloplasty alone (RA-r group). We analyzed treatment failure differences, the influence of residual MR, left ventricular remodeling, and clinical outcomes. The primary endpoint was treatment failure  within 5 years of follow-up after the procedure. Results. A total of 45 patients showed failure of the treatment within 5 years, of which 16 patients underwent SV-r + RA-r (35.6%) and 29 underwent RA-r (64.4%,  Ischemic mitral regurgitation (IMR) is a clinicopathological perturbance associated with significant morbidity and mortality ,2,3,4,5.The current guidelines still do not include specific recommendations on the use of subvalvular repair (SV-r) combined with restrictive annuloplasty repair (RA-r) in the treatment of IMR. This procedure has not been widely endorsed by the surgical community, and the most common surgical indications for SMR remain chordal-sparing mitral valve replacement (MVR) and restrictive annuloplasty ,14. The p < 0.001), maintaining the benefit achieved immediately postoperatively with freedom from major adverse cardiac and cerebrovascular events (p = 0.004) [In the papillary muscle approximation (PMA) randomized trial, 96 patients with severe chronic ischemic mitral regurgitation underwent complete surgical myocardial revascularization, associated with either isolated RA-r or SV-r plus RA-r, and were followed up on for 5 years. Left ventricular end-diastolic diameter improved at the 5-year follow-up  trial , and it We investigated long-term differences in treatment failure associated with a complete surgical myocardial revascularization, including the long-term influence of residual MV regurgitation, LV remodeling, and clinical outcomes, in the 96 patients with severe chronic IMR and coronary artery disease undergoing restrictive annuloplasty alongside subvalvular repair (SV-r + RA-r group) and restrictive annuloplasty alone (RA-r group).2, or by a combination of guideline-directed adjunctive echocardiographic quantification methods [SMR was assessed using transthoracic echocardiography (TTE) prior to the procedure. This was based on integrative criteria adjudicated by an independent echocardiographic core laboratory. SMR was defined as an effective regurgitant orifice area (EROA) of at least 0.4 cm methods . Coronar(1)Repositioning the PMs;(2)Standardized realignment of the subvalvular apparatus in one plane. This is achieved by the apico-lateral realignment of both PMs to eliminate apical tenting of the anterior leaflet;(3)Approximation of both PMs with the use of Goretex 4-0 stitch  for PMs for anatomy type 1–3 or with 4 mm Goretex graft  for PMs anatomy type 4–5.We developed and previously published research on the papillary muscle approximation technique ,4,11. BrCoronary artery bypass grafting (CABG) surgery was performed using standard techniques to achieve complete revascularization with the use of single or double internal thoracic arteries (ITAs). All patients received guideline-directed medical therapy for heart failure and coronary artery disease, including antiplatelet medication, neurohormonal antagonists, lipid-lowering agents, and cardiac resynchronization and defibrillator therapy as appropriate. Patients were followed for up to 5 years after the procedure.In detail, the subvalvular repair combined with restrictive mitral annuloplasty surgery was achieved using a median sternotomy under normothermic cardiopulmonary bypass conditions and intermittent anterograde blood cardioplegia. Before starting with the cardiopulmonary bypass, transesophageal echocardiography was performed to corroborate that any mitral valve structural abnormalities occurred and all eligibility criteria were met.End-diastolic and end-systolic interpapillary muscle distances (IPMD) were compared with preoperative values measured via transthoracic echocardiography in the parasternal short-axis view of the LV. The comparison of IPMD in physiological and pathological conditions represents a fundamental point for the success of the procedure. The interpapillary muscle distance measured in mid-systole was between 12.9 mm and 22.5 mm. It is important to highlight that these measures show a fluctuation when normalized to BSA and corrected with age. The measure of IPMD normalized to BSA and age is 10.5 mm more or 3.3 mm less .To achieve optimal exposure of the MV through the left atriotomy, we used the Carpentier retractor . At the time of the surgical inspection, all patients experienced typical lesions attributable to the Carpentier type IIIb classification that confirmed the echocardiographic diagnosis. The geometrical disorder of MV was characterized by changes to three measures: anteroposterior annular dilation, tenting area, and interpapillary muscle distance. In all patients, we noticed leaflet restriction as the result of excess traction on the leaflets, leading to a lack of coaptation.The first step, after diastolic cardioplegic arrest, was to recognize and carefully inspect the PMs intraoperatively. IPMD was measured with a flax thread to confirm the echocardiographic findings. Variations in the measured PM distance decreased, and a second measurement was performed by the same method to corroborate the result. It is important to note that PMs which were identified anatomically as type I and II were approximated with a CV-4 Gore-Tex suture . The suture was arranged on the head of each PM . ConsideParticular attention was paid to the evaluation of the chordal organization to optimize the correction of regurgitation, for which the exact anatomical location of the tendon cords was evaluated. Usually, the chordae originate from the PPM and insert on the scallops P2 and P3 of the posterior leaflet; however, chordae from APM are attached to the anterior leaflet, which leads to the development of the ‘‘seagull sign’’ and respective tenting.The second phase was the calibration of the anterior leaflet area to perform the RMA. Then, the AL was measured using a prosthetic ring obturator. Annuloplasty was undersized by 2 sizes and was performed with a Carpentier-Edwards Physio ring . The prosthetic ring was fixed with the use of 2/0 braided sutures placed 1 mm away from the leaflet’s hinge on the atrial wall. Sutures were positioned circumferentially, starting from the posterior commissural area, in counterclockwise fashion. Larger bites were used at the posterior area of the annulus from trigone to trigone to accentuate the downsizing effect at this level. All sutures were then passed through the prosthetic ring cuff. The ring was lowered into position and the sutures were tied.The third phase was the concomitant CABG operation, which was performed on all patients. We used a single or double internal thoracic artery (ITA) for complete revascularization of diseased coronary arteries. In patients less than 65 years of age, we preferred the use of both Y-shaped and in situ RITA and LITA.The endpoints of this study differ from previous studies ,4,5 and The primary endpoint was treatment failure: this was defined as a composite of death, MV reoperation, or recurrence of moderate-to-severe (3+) or severe (4+) MR within 5 years after the procedure.Secondary endpoints included all-cause death at 30 days and within 5 years of follow-up; the echocardiographic degree of MR at discharge and 6 months, 1 year, 2 years, and 5 years; left ventricular reverse remodeling on basis of transthoracic echocardiography with particular regard to the left ventricular ejection fraction (LVEF); and the left ventricular end-systolic dimension and the functional status as assessed by the New York Heart Association (NYHA) class related to the progression of residual MR at follow-up. MR was graded according to the recommendations of the European Association of Cardiovascular Imaging using an integrative approach, which included qualitative, semiquantitative, and quantitative measures .Echocardiographic endpoints, determined by by transthoracic echocardiography (TTE) examination, included the severity of the LV disease and MV tenting indicators , which were determined at end-systole in the parasternal long-axis view. Tenting height (TH) was then measured as the distance between the mitral annular plane and the most atrial margin of the coaptation zone. The two-dimensional tenting area was fixed as the area between the mitral annular plane and the mitral leaflets at end-systole. Coaptation length was interpreted as the length of the overlap between the anterior and posterior mitral leaflet during systole. The annular plane was defined as the junction between the anterior and posterior hinge points of the mitral leaflets. Other parameters of severity were tenting angles, which were measured between the mitral leaflets, and the mitral annular plane alongside the interpapillary muscle distance, both of which were determined in the parasternal short-axis view of the left ventricle.t-tests were used for normally distributed data, which are presented as mean ± standard deviation in text and tables. Incidence rate (IR) differences (IRD) were calculated for the MR and NYHA functional classes. Event-free survival curves were constructed using the Kaplan–Meier method, and treatment effects were estimated as hazard ratios with a 95% confidence interval, which was derived using the Cox proportional hazards model. The proportional hazards assumption was tested with the use of a graphical method. A multivariable adjustment was further performed with baseline age, gender, diabetes, LVEF, and severity of MR in view of the presumed association with treatment failure. Similarly, a multivariable adjustment with baseline age, gender, diabetes, and LVEF was performed to test the impact of moderate or moderate-to-severe residual MR on mortality at follow-up. A p-value < 0.05 was considered statistically significant. The analyses were conducted using R statistical software  through the survival, survminer, and subtee packages.Categorical data are displayed as frequencies and percentages in text and tables, with Fisher’s exact test or the chi-square test used as appropriate. Normality criteria were checked and met for each continuous variable. Independent sample or paired sample In total, 96 patients underwent randomization, 48 receiving subvalvular repair with restrictive annuloplasty repair (SV-r + RA-r group) and 48 receiving restrictive annuloplasty repair alone (RA-r group). The average native annulus size was 39.1 mm in patients who received the SV-r + RA-r and 39.5 mm in patients who received RA-r alone. The ring sizes used were 26 mm (42.7% of patients) and 28 mm (57.3% of patients). Concomitant CABG was performed in 100% of patients. There were no crossovers between the groups post-randomization. Patients underwent clinical and echocardiographic follow-ups at 6 months, 1 year, 2 years, and 5 years after the surgical procedure. The follow-ups were 100% complete. Baseline patients’ characteristics and procedural details have previously been reported [p = 0.006, p = 0.02; HR adjusted 0.48, 95% CI: 0.24–0.93, p = 0.03, Within 5 years, 45/96 patients (46.9%) presented with treatment failure. Of these, 16 patients underwent SV-r + RA-r (35.6%) and 29 underwent RA-r . Within 5 years after surgery, 1 patient (7%) underwent MV reoperation in the SV-r + RA-r group, and 5 (21.7%) in the RA-r group (p = 0.09).The overall survival rates were 92% (95% CI: 88–98%) and 74% (95% CI: 66–83%) at 30 days and 5 years, respectively. Survival at 5 years was 77% (95% CI: 66–90%) in the SV-r + RA-r group and 71% (95% CI: 59–85%) in the RA-r group (p = 0.36), gender (p = 0.6), diabetes (p = 0.98), baseline LVEF (p = 0.42), or baseline severity of MR (p = 0.93).Subgroup analysis showed that the association of the treatment strategy with treatment failure was consistent across all the examined subgroups. There were no significant interactions between the impact of SV-r and ± RA-r on treatment failure within 5 years of follow-up and age  had no (0) or mild (1+) residual MR at discharge. At 5 years, a moderate-to-severe (3+/4+) residual MR was observed in 5% in SV-r + RA-r versus 32% in RA-r group  and in the RA-r group  compared to the first echocardiographic evaluation at 6 months after the surgery.As shown in p < 0.0001). At 5 years, the IRs of NYHA functional classes III and IV were significantly lower (1.6% vs. 16.5% patients per year) in the SV-r + RA-r group vs. the RA-r group . NYHA functional class III or IV was reported in 3 patients (8.1%) of the SV-r + RA-r group and 28 patients (82.3%) of the RA-r group  than those with no or 1+ MR  after adjusting for key covariates associated with each outcome of interest, restrictive annuloplasty repair remained at an increased risk of failure and mortality at 5 years compared to subvalvular repair combined with restrictive mitral annuloplasty, and the rates of recurrent mitral regurgitation were higher and occurred earlier in the RA-r group than in the SV-r + RA-r group; (2) although functional status improved in both groups after mitral valve surgery, at 5 years, the RA-r group had worse health-related and heart failure-related outcomes post-operatively than the SV-r + RA-r group; (3) LVESD, a surrogate for LV reverse remodeling, improved significantly in patients who received subvalvular repair and restrictive mitral annuloplasty compared to those who received restrictive mitral annuloplasty alone after 5 years; (4) the improvement of left ventricular remodeling is closely correlated with mitral regurgitation recurrence. The worsening of ventricular dimensions was significantly higher in patients who developed earlier 2+ or 3+ MR.p = 0.006).In the present analysis, we found that, following adjustment for baseline confounders, restrictive annuloplasty repair was associated with higher treatment failure and risk of mortality at 5 years after mitral valve surgery. The reasons for the inferior outcomes of restrictive annuloplasty repair after mitral valve surgery for secondary mitral regurgitation are clearly attributable to differences in left ventricular remodeling (measured with changes in LVESD) and to the differential grade of MR recurrence during follow-up on patients who underwent subvalvular repair plus restrictive annuloplasty compared to those who received restrictive annuloplasty repair alone. However, more importantly, in the present analysis, the absolute differences between treatment failure, recurrent MR, and death with SV-r ± RA-r appeared to be higher in the group who underwent RA-r (p = 0.007).In our analysis, we observed that 32% of patients in the RA-r group experienced grade 3+ MR recurrence, compared to 5% in the SV-r + RA-r group. No patients had severe mitral regurgitation in the SV-r + RA-r group. These findings were seen in the RA-r group, for whom the duration of mitral repair was shortened due to recurrent mitral regurgitation. This may confer a predisposition to heart failure, atrial fibrillation, and repeat surgery or hospitalization. We noted that patients who received restrictive mitral annuloplasty had more rapid progression to 2+ or 3+ recurrent MR than patients who received associated subvalvular repair, the majority of whom developed moderate recurrent MR only in the fifth year of follow-up . Although LVESD improved significantly from the baseline in the two groups during the first year after surgery, we observed only stable improvement from baseline over the five years of follow-up in patients in the SV-r plus RA-r group. We also suggested that the worsening of ventricular dimensions was higher in the patients who developed residual MR 3+/4+, as demonstrated in the RA-r group (p = 0.007).In the present analysis, we observed estimated lower incidence rates of NYHA functional class III or IV (1.6% vs. 16.5% patient-years) in patients undergoing subvalvular repair plus restrictive mitral annuloplasty compared with those undergoing only restrictive mitral annuloplasty  reported that mitral valve replacement was not associated with significant differences in left ventricular remodeling at 1- and 2-year follow-ups when compared with restrictive annuloplasty ,9.Indeed, in the CTSN trial, although the left ventricular end-systolic volume index (LVESVI) improved significantly from the baseline in the two groups during the first year after the mitral valve procedure, only a small improvement was observed during the second year .2 vs. 63 ± 27 mL/m2), and, surprisingly, compared to those patients who had undergone mitral valve replacement (61 ± 39 mL/m2) [Importantly, a sub-analysis of the CTSN study provided an intriguing case in point. In this trial, 74 patients with severe IMR who did not have persistent or recurrent mitral regurgitation after restrictive mitral annuloplasty had significantly smaller left ventricles at the 2-year follow-up as compared to those patients with recurrent MR after RA alone  [p = 0.356 and p = 0.49 respectively) [2 and LVEDD > 60 mm) [The CTSN trial underwent some criticism because 26.2% of patients who received restrictive mitral annuloplasty did not have concomitant coronary surgical revascularization . As it w> 60 mm) ,28,29.Besides the previously described limitations to the primary analysis ,4, it shFinally, although the echocardiographic controls were performed by two blinded expert cardiologists, guaranteeing the reliability of the results, for the wall motion abnormalities, they were classified as inferior, infero-posterior, or anterolateral. The conventional 16-segment division used in transthoracic echocardiography would have required a much larger sample size, which was too many participants for this study to consider it as a primary endpoint.SMR is a pathology that impairs the geometrical relationship between the ventricle and the MV apparatus. Reducing the size of the annulus alone does not achieve durable results over time, and results in an increased incidence of treatment failure as the composite endpoint, rehospitalization, and reintervention, primarily due to early MR recurrence. The addition of subvalvular repair improves the durability of the repair, thus extending all the benefits of preventing MR recurrence and offering a sustained improvement in echocardiographic and clinical parameters over time. Worsening MR recurrence heralds worsening LV parameters in both groups. Large-scale multicenter trials are encouraged to confirm the benefits of double-level repair as part of the armamentarium of IRM management, and to support its indication in future guidelines.


The ISO (n = 14) group only performed isometric squats, and the control group  performed a 5-min treadmill run at 6 km/h. The countermovement jump (CMJ) height results were analyzed from the baseline and then at 3, 5, 7, and 9 min after the PAPE protocols. The statistical significance was set at p < 0.05. RM-ANOVA revealed differences in the group-minute interaction , and post-hoc tests demonstrated a significant decrease in CMJ after 5 min in the ISO group (p < 0.0446). The performance of the ST-ISO group markedly decreased in the 3rd and 7th min after PAPE , though it improved significantly in the final minute (p < 0.0030). Chi-squared analysis revealed that the ST-ISO group peaked more frequently in the 9th min . However, CMJ height improvement did not differ between the PAPE protocols, thus it was close to statistical significance . The ST-ISO protocol provided jump enhancement, though the deterioration observed in the first minutes after the protocols suggest the rest period after activity requires attention, and the methods need to be individualized.This study aimed to establish the effectiveness of slow tempo bodyweight squat combined with an isometric squat (ST-ISO), and an isometric squat alone (ISO), as a post-activation performance enhancement protocol (PAPE) for jump height improvement. The study sample consisted of 41 trained men aged 18–24. The ST-ISO group ( Jump height is a reliable indicator of athletic performance affected by power output a body mass or force-velocity profile . Jump heThe phenomenon called post-activation performance enhancement (PAPE) makes it possible to evoke immediate, temporal improvement in physical performance . The PAPThe literature demonstrates the efficiency of interventions based on isometric efforts with external load  and voliTypically, in resistance training programming as well in PAPE protocols creation primarily are consider intensity (load), volume and rest break . Lastly Using voluntary isometric contractions and slow movement tempo has a low risk of injury due to a low external load and does not require special equipment what also make it is easy to implement in physical effort preparation . HoweverThe Ethics Committee of the Wroclaw University of Sport Health Sciences approved this study (6/2023), which followed the guidelines of the Declaration of Helsinki.This study was a parallel randomized controlled trial. Firstly, volunteers were asked to fill the questionnaire and provide data: age, injury history in last 8 weeks, training experience, use of steroids, and back squat best in last 4 weeks. Then, they were meet on the familiarization session where basic morphological measurements were performed countermovement jump (CMJ) technique was introduced and slow tempo bodyweight squat (5-0-5-0). Then, bar height used for the PAPE protocols was individualized by participants performing a half-squat with a knee angle of 90°. As such, the height of the immovable bar was adjusted based on pushing with maximum effort from a pre-established position. Also, the back squat strength was verified, the participants were asked to perform the at least one repetition with 120% body weight.www.randomizer.org) tool, including an ST-ISO group, ISO group, and control group (CG). Simple random sampling without replacement was used. The sampling frame was a list of participants in alphabetical order and coded by number 1–56.Participants were stratified into one of three groups using the Research Randomizer  and injury (n = 4). No one declare using steroids or experience in weightlifting tournaments. Therefore, in the main stage, 56 subjects were qualified, but one participant from the ST-ISO, four from ISO, and seven from the control group left the study during the experiment and did not complete all measurements—finally, data from 41 subjects  were consider. A detailed description of the participants was presented in the section results.The initial assessment identified 64 male subjects as potential study participants recruited from members of gym and fitness clubs’ members. The inclusion criteria were an age of 18–24 years old, no musculoskeletal injury 8 weeks before the study, no other medical contradictions, experience in strength training of at least 6 years and at least 3 years in continuous resistance training, the ability to perform a back squat with 120% of body weight, whereas exclusion criteria was medical contradiction, using doping substances such as anabolic-androgenic steroids, experience in weightlifting tournaments or actually being professional athlete in any sport discipline, not being able to perform full back squat with 120% bodyweight load. Application of the inclusion criteria led to the exclusion of subjects based on age , and body mass was assessed using an InBody230 device , which has been confirmed to be highly reliable .2/8). There were no CMJ depth restrictions, and participants had to keep their arms on their hips and couldn’t swing their arms. The validity and reliability of the OptoJump device in jump height measurements were previously confirmed  measured CMJ height at a frequency of 1,000 Hz using the equation: (9.81 × (flight time)onfirmed .α = 0.05, the sample size required to detect changes in the measured parameters was 39 subjects.Assuming 95% power and a minimum effect size (ES) of 0.20 at The CMJ measurements were validated for test-retest reliability using the intraclass correlation coefficient (ICC 2.1) criteria of post-hoc test least significant difference were performed to detect detailed differences between groups. Meanwhile, a chi-squared analysis assessed differences between groups in time-to-achieve peak performance and the frequency of positive protocol responses. Student’s t-test for independent samples compared incremental jump height changes between groups. In addition, Cohen’s d values for ES were calculated  was used for the analysis.The results were expressed as mean, standard deviation (SD) and 95% confidence intervals (95% CI). The Shapiro–Wilk test examined the data distribution, while Levene’s confirmed the homogeneity of variance and Mauchly test was performed to assess data sphericity. A one-way analysis of variance (ANOVA) examined differences in baseline measurements between groups, and a two-way repeated-measures ANOVA (RM-ANOVA) compared differences between groups and time and lculated . The sigThe ICC 2.1 for CMJ measurements equated to 0.95, and the CV was 0.1, confirming the measurements’ reliability.In the next of the analysis the jump height results from consecutive minutes provided in p = 0.8484, η2 = 0.0085) though did it for time  and group-time interaction .Two-way RM-ANOVA (group (3) × time (5)) revealed no statistically significant differences for the group . In this regard, CMJ height decreased in the 5th min and returned to a higher level in the 9th min. In the ST-ISO group, CMJ height decreased in the 3rd (p = 0.0137) and 7th (p = 0.0424) minutes compared to the baseline. Performance peaked in the 9th min, with an increase observed over the baseline ((p = 0.0030) 3rd (p < 0.0001) 5th (p = 0.0008) and 7th (p < 0.0001) minutes). In addition, performance decreased to a greater extent in the 3rd min than in the 5th min (p = 0.0384)  .2 = 17.97, p = 0.0214), which, along with the peak performance observed in the 9th min, suggests ST-ISO protocol-related improvement. Also, analysis of the frequency of positive responses to the PAPE protocol revealed that both interventions provided positive reactions compared to the control group .The chi-squared analysis revealed a statistically significant association between the time and individual peak performance (Xp = 0.0781), with an ES of 0.7.In the final analysis, changes in the best results from baseline were comparable between both PAPE protocols. The differences between ISO (0.9 ± 1.2) and ST-ISO (1.3 ± 1.1) were close but insignificant (t = −1.82, This study aimed to describe and compare the efficacy of two PAPE protocols based on volitional maximal isometric effort, with a slow-tempo full-squat added to one of the procedures. Generally, both procedures produced similar effects whereas ST-ISO protocol trend to bring more remarkable improvement. However, observation presented some level of uncertainty due elevated range of noted results therefore caution is needed. Despite, deterioration was visible in the first-minute post-intervention, which suggests a requirement for an extended rest period. Indeed, jump height decreased below the baseline in the ST-ISO group, although it improved significantly 1 min after the protocol. Additionally, some ambiguity added jump height fluctuation where decrease and increase occur alternately. The higher decrease during the initial post-protocol period may be related to the higher load associated with the additional slow-tempo squats. On the other hand, the improvement observed after 9 min indicates that the protocol led to appropriate neuromuscular stimulation and evoked a PAPE effect. Moreover, this suggests a relationship between load and rest interval, and an extended break is required when using a higher load.Using the back squat as a form of conditioning activity to evoke jump height improvement is an effective tool, but avoiding fatigue is crucial . TherefoUsing isometric effort-based PAPE protocols effectively evokes athletic performance improvement . Some obIsometry has the potential to be combined with other types of activity, with The study was limited by not using a force platform or dynamometer to control the force output of the participants, both of which inform on the stimulus magnitude during voluntary isometry. It also made enable to directly measures power and force during the jump measures and analyzed these parameters. Also, post-activation CMJ height measurements did not extend beyond the time of peak performance in the ST-ISO group. Only males’ sample was examined, therefore is need to investigate the effects in females. Future research should address these limitations.Both protocols led to a deterioration in performance in the initial post-activation minutes, with a more remarkable decrease in the ST-ISO group and a subtler decrease in the ISO group. However, the ST-ISO group improved in the following minutes and peaked at the final measurement time, while the ISO group returned to baseline levels. Nonetheless, decreased CMJ height magnitude indicated that ST-ISO increased fatigue more than ISO. The significant improvements recorded in the minutes following this decrease suggest that prolonging the rest period by at least 9 min should achieve better results. However, when compare absolute difference of improvement between both protocol ST-ISO trend to be more effective in jump height enhancement than ISO. The results of this study revealed some positive effects of combining slow movement bodyweight squats with isometric activity. However, caution is demanded when interpreting these results due to some ambiguities associated with the observed alternating pattern of jump height decrease and increase, also observed results are characterized by quite wide range. Therefore, further studies are necessary to extend the observation time and include a larger sample size, which could allow for explaining the observed differences in more detail.10.7717/peerj.15753/supp-1Supplemental Information 1Click here for additional data file.


GNAS gene, which encodes the alpha subunit of G proteins, leading to constitutive activation of several G Protein-Coupled Receptors (GPCRs). The co-occurrence of two of the above-mentioned cardinal clinical manifestations sets the diagnosis at the clinical level. In this case report, we describe a 27-month-old girl who presented with gonadotropin-independent precocious puberty secondary to an estrogen-secreting ovarian cyst, a café au lait skin macule and growth hormone, and prolactin excess, and we provide an updated review of the scientific literature on the clinical features, diagnostic work-up, and therapeutic management of MAS.McCune–Albright syndrome (MAS) is a rare sporadic condition defined by the classic triad of fibrous dysplasia of bone, café au lait skin macules, and hyperfunctioning endocrinopathies. The molecular basis of MAS has been ascribed to the post-zygotic somatic gain-of-function mutations in the  GNAS gene , which encodes the alpha subunit of G proteins, in affected tissues from all the patients of the study, as well as in tissues not typically involved in the syndrome [GNAS is responsible for the clinical manifestations of the syndrome in the majority of the cases (over 95%) [McCune–Albright syndrome (MAS) is a rare, complex non-hereditable genetic disorder, which is characterized by a variable combination of fibrous dysplasia of bone, café au lait pigmented skin lesions, and hyperfunctioning endocrinopathies . This sysyndrome . Interessyndrome . Since tver 95%) . Rarely,ver 95%) . All thever 95%) . Althougver 95%) .MAS is a rare sporadic disease with an estimated prevalence between 1/100,000 and 1/1,000,000 . The carThe therapeutic management of patients with MAS is only symptomatic and requires a multidisciplinary team with experience in pediatrics, endocrinology, orthopedics, pain management, and rehabilitation . Girls wIn this case report, we describe a 27-month-old Greek girl with vaginal bleeding due to an ovarian cyst of the right ovary leading to gonadotropin-independent precocious puberty, a café au lait skin macule, and growth hormone excess, indicative of the diagnosis of MAS. This study was reviewed and approved by the “Aghia Sophia” Children’s Hospital Committee on the Ethics of Human Research, approval number 1707-01/02/2022. Written informed consent was obtained from the parents of the patient for the publication of this case report and any accompanying images. All data generated or analyzed during this case report are included.A Greek girl presented at the age of 27 months with vaginal bleeding lasting three days. Mucous vaginal discharge without signs of bleeding had been noted seven days before. The girl was born after normal delivery at full term and was the first child of non-consanguineous parents. She weighed 3550 gr (50th centile) with a length of 50 cm (50th–75th centile) at birth and had no gross congenital anomalies. There was no past medical history and no history of medications. The mother had developed gestational diabetes which was managed with dietary measures.Upon admission at the hospital, an abdominal ultrasound was performed which showed an ovarian cyst of the right ovary measuring 5 cm × 4.1 cm × 3.5 cm with clear content and a thin layer. The uterus was found to be of the infantile type with a thickened secretory endometrium (of 1 cm thickness) and a thickened vagina. No adrenal pathology was found.The girl was initially admitted to the pediatric surgery department for 2 days. Laboratory investigations revealed increased levels of estradiol (111.9 pg/mL), prolactin ) and growth hormone (26.5 ng/mL) with both random blood FSH and LH levels ˂ 0.1 mUI/mL and normal IGF-1 (122 ng/mL (18–172 ng/mL)).The girl was subsequently transferred to the pediatric department. Careful systemic clinical evaluation revealed a café-au-lait skin macule with jagged borders measuring 2 cm × 2.5 cm on the lower back which respected the midline. Coarse facial features and hyperpigmented labia minora were also noted. The child’s weight, height, and head circumference were along the 90th–97th centile, 50th–75th centile, and 75th–90th centile, respectively . Breast A further endocrine work-up was then performed. The levels of testosterone, dehydroepiandrosterone sulfate (DHEAS), and androstenedione were found to be within normal reference range: testosterone ˂20 ng/dL, androstenedione: ˂0.3 ng/mL, DHEAS: ˂15 μg/dL. CA 125, CA 19-9, and β-HCG were also within normal range: CA 125: 15.4 U/mL , CA 19-9: 31.5 U/mL , and β-HCG: 0.1 mIU/mL). Due to the clinical and laboratory indications of GH-excess, an oral glucose tolerance test was performed which demonstrated no suppression of GH (nadir GH: 5.67 ng/mL at 120 min). Subsequently, a brain and pituitary MRI was performed which revealed no abnormalities.The adrenal evaluation showed normal ACTH (44.99 pg/mL) and cortisol (21.90 μg/dL) concentrations. Measurements of TSH (3.25 μUI/mL) and fT4 (1.19 ng/dL) concentrations were within normal range, and the thyroid ultrasound depicted no abnormal findings.The MRI of the lower abdomen and pelvis revealed a cystic lesion measuring 3.94 × 3.85 × 2.26 cm in diameter without features of a benign or malignant tumor.A cardiac echocardiogram was performed and showed no pathology.Based on the clinical and laboratory presentation of gonadotropin-independent estradiol production resulting in precocious puberty, the ovarian cyst, the laboratory evidence of growth-hormone excess along with the presence of the café-au-lait skin macule with characteristic features of irregular borders (Coast of Maine) and a distribution showing the so-called “respect of” the midline of the body, a clinical diagnosis of McCune–Albright Syndrome was made. The girl was then evaluated for additional features of McCune–Albright Syndrome with an assessment of the skeletal system: a thorough skeletal survey was performed  which showed no signs of fibrous dysplasia.The girl was then discharged with a plan for follow-up by a multidisciplinary team consisting of Pediatricians, Endocrinologists and Orthopedics. Due to evidence that bone lesions may develop over time, the skeletal assessment was recommended to be repeated in the future. However, shortly after discharge, due to the recurrence of vaginal bleeding and parental anxiety, the decision was made to start treatment of precocious puberty with an aromatase inhibitor. At that time, the patient did not receive any medication for growth hormone and prolactin hypersecretion. As the family moved away from Greece, further follow-up was planned to occur abroad in another pediatric endocrine department.In our case report, the 27-month-old girl presented with a 3-day history of painless vaginal bleeding and was first admitted to the pediatric surgery department in “Aghia Sophia” Children’s Hospital, Athens, Greece. Upon admission, a physical examination revealed no signs of sexual abuse, trauma, or foreign body, while the initial laboratory investigations did not show thrombocytopenia, coagulation disorders, or infection. The abdominal ultrasound depicted a large ovarian cyst of the right ovary with clear content and a thin layer, excluding the possibility of a benign or malignant tumor. Moreover, CA 125 and CA 19-9 measurements were within a normal range, further excluding malignancy from the differential diagnosis of vaginal bleeding. Importantly, endocrinologic evaluation revealed high estradiol concentrations with both suppressed FSH and LH, indicating that the ovarian cyst was autonomously estrogen-producing, and suggesting gonadotropin-independent precocious puberty . Indeed,GNAS mutation(s) because the mutation detection rates in PCR-based sequencing methods are approximately 20–30% in peripheral blood lymphocytes and >80% in affected tissues [GNAS mutations are the level of mosaicism in the tested tissue and the sensitivity of the method [GNAS mutations in patients with MAS [The diagnosis of MAS was set clinically in this girl. The presence of a café-au-lait skin macule with irregular borders (Coast of Maine) and a distribution respective of the midline of the body, in association with gonadotropin-independent precocious puberty and growth hormone excess fulfilled two of the cardinal criteria of MAS diagnosis . We did  tissues ,32 and te method ,29. Intewith MAS ,34,35,36The patient was first admitted to the pediatric surgery department in “Aghia Sophia” Children’s Hospital. The detailed physical examination (café-au-lait skin spot and breast development II according to Tanner staging), as well as the initial endocrinologic evaluation (gonadotropin-independent precocious puberty secondary to autonomous estrogen-secreting ovarian cyst), were indicative of the diagnosis of MAS preventing any surgical management. Papanikolaou and Michala have reviewed the published literature about current management for autonomous ovarian cysts in prepubertal girls and concluded that a conservative approach should always be the first-line choice, taking into consideration the high risk of recurrence in cases of MAS . Undoubtsine qua non to achieve the best therapeutic outcomes.The co-occurrence of gonadotropin-independent precocious puberty and growth hormone excess in our patient is rare in cases with MAS . Both thIn summary, we described a 27-month-old Greek girl with gonadotropin-independent precocious puberty secondary to an estrogen-producing ovarian cyst, a café au lait skin macule and growth hormone excess, all suggestive of MAS diagnosis and highlight the importance of careful assessment and diagnostic work-out in order to prevent unnecessary or even dangerous surgical approach.


SERPINE1, FEN1, PDGFRB, SNCG, TCF3, and APOC3. The TCGA-STAD cohort was utilized as a training set while the GSE84437 and GSE13861 cohorts were analyzed for validation. Immune cell infiltration and immunotherapy efficacy were investigated in the PRJEB25780 cohort. Data from the genomics of drug sensitivity in cancer (GDSC) database revealed pharmacological response. The GSE13861 and GSE54129 cohorts, single-cell dataset GSE134520, and The Human Protein Atlas (THPA) database were utilized for localization of the key senescence-related genes. Association of a higher risk-score with worse overall survival (OS) was identified in the training cohort  and the validation cohorts . The risk-score was positively correlated with densities of tumor-infiltrating immunosuppressive cells (P < 0.05) and was lower in patients who responded to pembrolizumab monotherapy (P = 0.03). Besides, patients with a high risk-score had higher sensitivities to the inhibitors against the PI3K-mTOR and angiogenesis (P < 0.05). Expression analysis verified the promoting roles of FEN1, PDGFRB, SERPINE1, and TCF3, and the suppressing roles of APOC3 and SNCG in GC, respectively. Immunohistochemistry staining and single-cell analysis revealed their location and potential origins. Taken together, the senescence gene-based model may potentially change the management of GC by enabling risk stratification and predicting response to systemic therapy.As one of the prevalent tumors worldwide, gastric cancer (GC) has obtained sufficient attention in its clinical management and prognostic stratification. Senescence-related genes are involved in the tumorigenesis and progression of GC. A machine learning algorithm-based prognostic signature was developed from six senescence-related genes including  Gastric cancer is one of the most prevalent tumors, with the fifth-highest incidence and fourth-highest mortality rate all over the world . ExploriG12VHRAS is usually associated with upregulated senescence-related genes including p53, ARFp19, INK4a, Pmlp16, and retinoblastoma, which work as an obstructive factor for tumor initiation [In mammalian cells, stimulated oncogenes accompanied by inactivated tumor-suppressor genes (TSGs) are crucial inducements of proliferative stress and induction of cellular senescence, which therefore limit tumor growth –5. For iitiation . Howeveritiation , 9.Senescence-related secretory phenotype (SASP) refers to the ability of senescent tumor cells to actively produce a wide variety of proteins, many of which are pro-inflammatory cytokines or pro-inflammatory substances in themselves , 11. SASHere, based on senescence-related genes, we sought to develop a model for the prognostic stratification of GC. A favorable prognosis was observed in the low-risk group, together with low sensitivities to the inhibitors against the PI3K-mTOR and angiogenesis, low densities of immunosuppressive tumor-infiltrating immune cells, and a high response rate to pembrolizumab monotherapy.TP53 was the highest (46%) followed by PIK3CA  pathway analysis, these DEGs were mainly enriched in cell cycle regulation, homologous recombination, base excision repair, and P53 pathway  = 1.93; 95% confidence interval (95% CI), 1.38–2.71; FEN1 exhibited improved OS  as continuous variables in the TCGA-STAD cohort (SERPINE1) + (0.120 × APOC3) + (0.090 × SNCG) + (0.015 × PDGFRB) – (0.128 × TCF3) – (0.133 × FEN1). Assigned with a risk-score, patients were stratified into high- or low-risk groups by the median value in the cohort. Patients in the high-risk group had higher expression of SERPINE1, APOC3, PDGFRB, and SNCG and lower expression of FEN1 and TCF3 , together with worse OS , resting memory CD4+ T cell , naïve B cells , and resting mast cells . Consistent with the risk-score, expression of SERPINE1, PDGFRB, and SNCG were also positively associated with M2 macrophage, resting memory CD4+ T cell, and naïve B cells, while expression of FEN1 and TCF3 were negatively associated with M2 macrophage, resting memory CD4+ T cell, and resting mast cells  staining revealed the distribution of the 6 senescence-based risk-score-related genes in GC. According to the single-cell RNA sequencing profile of the GSE134520 dataset, a total of 9 cell types were identified . APOC3 wt cells. –4H.Studies about GC, one of the most prevalent gastrointestinal (GI) malignancies, have increasingly concentrated on the prognostic implications of several signatures , 18. BasIn earlier research, prognostic models for GC patients were developed using sequencing data and clinicopathologic indicators –22. ClinThe biological features of GC may aid in predicting which tumors will benefit from chemotherapy and other targeted agents . CompareFurther, the drug sensitivity analyses add evidence for our model’s association with cancer and its potential clinical application. The PI3K-mTOR signaling pathway plays an important role in cancers and its inhibitors have shown efficacy in clinical trials . The PI3in vitro and in vivo experimental validation were highly recommended to examine the significance of the risk-score in GC and other cancers. Besides, further promising studies are recommended to explore the linkage between the 6 senescence-related genes and response to chemotherapeutic and targeted chemicals in animal models.As for limitations, the retrospective nature of this study has determined the limited capacity of the model, and prospective validation in well-designed cohorts is required to demonstrate its clinical value. Despite the consistent results among the survival analysis of TCGA and GEO cohorts, gene expression levels, IHC staining, and single-cell sequencing results, In summary, a novel and robust prognostic model consisting of six senescence-related genes was developed and validated in patients with GC. Additionally, the score of this model was associated with TIME and responses to chemotherapeutic, targeted, and immunotherapeutic therapies. The senescence gene-based model can potentially change the management of GC by enabling risk stratification and predicting response to systemic therapy.The transcriptomic, genomic, and survival data of 348 GC and 31 controls were retrieved from the TCGA-STAD (Data Release 31.0) cohort database. The transcriptomic and clinical data of 431 GC samples and 45 GC patients treated with pembrolizumab monotherapy were collected from the GSE84437 and the PRJEB25780, respectively. The expression matrix of tumor and normal tissues from the GSE54129 cohort  and the GSE13861 cohort , respectively. The single-cell RNA sequencing data of 13 fresh human tissue samples from nine GC patients were retrieved from the GSE134520 dataset. The process and specific cohorts used in the analysis were depicted in The gene expression profiles were analyzed in tumorous and normal samples of the TCGA-STAD cohort via the “limma” R package (Version 3.28.14), and genes with a false discovery rate (FDR) <0.05 were selected as DEGs. Gene ontology (GO) and KEGG analysis were conducted for functional enrichment of the DEGs utilizing the “clusterProfiler” R package (Version 3.0.4).2 (Fragments Per Kilobase Million + 1) value of each gene. The LASSO method  was utilized for model construction in the training set (the TCGA-STAD cohort) based on the DEGs with a significant association with OS [The prognostic value of every single senescence-related gene was examined by univariable cox regression using the R package “survival” according to the log with OS . The numn” depicted the number of genes involved in the model, while“expi” and “βi” represents the mRNA level and regression coefficient of gene i, respectively.“Assigned with a risk-score, patients were stratified into high- or low-risk groups by the median value in the cohort. The R package “survival” (version 3.4.0) was applied for survival analysis comparing the OS between the high- and low-risk groups. The prognostic value of the model was evaluated with the AUC and C-index values, and visualized by the receiver operating characteristic (ROC) curve by the “timeROC” R package (Version 0.4). The GSE84437 and the GSE13861 cohorts were utilized for validation.Based on the cell types categorized by the deconvolution approach in CIBERSORT , the denhttps://www.cancerrxgene.org), we calculated the correlations  of the half maximal inhibitory concentration (IC50) with the mRNA expression and the risk-score. The results are obtained by the “pRRophetic” (Version 4.0.2) and the “ggplot2” (Version 3.3.6) R packages. P values were adjusted by the FDR method.We analyzed the response to pembrolizumab monotherapy in GC patients from the PRJEB25780 cohort. Based on the genomics of drug sensitivities in cancer (GDSC) database (https://www.proteinatlas.org/).The mRNA levels of the 6 genes involved in the signature were compared between GC tumors and normal tissues from two more cohorts , together with the expression levels compared among different cell types in GC tissues from the GSE134520 dataset, with annotation from the Tumor Immune Single-cell Hub (TISCH) database for cell identification. IHC staining figures in GC tissues and normal gastric samples were obtained from The Human Protein Atlas database . If not stated above, P < 0.05 illustrated statistical significance.Statistical results generated in this study were conducted in R (Version 3.6.0), SPSS (Version 23.0), and GraphPad Prism (Version 8). Wilcox test was used to analyze the association between the senescence-related gene signature and immune characteristics. Survival analyses were conducted by the Log-rank test, with visualization by the Kaplan-Meier (KM) curves. The independence of the prognostic signature was verified by univariable and multivariable Cox regression, with the input of significant variables into the multivariable analysis by Supplementary Figure 1Supplementary Tables


In July 2020, Mayo Clinic launched Advanced Care at Home (ACH), a high-acuity virtual hybrid hospital-at-home model (HaH) of care at Mayo Clinic Florida and Northwest Wisconsin, an urban destination medical center and a rural community practice respectively. This study aims to describe demographic characteristics of ACH patients as well as their acuity of illness using severity of illness (SOI) and risk of mortality (ROM), to illustrate the complexity of patients in the program, taking into account the different diagnostic related groups.Mayo Clinic uses All Patient Refined-Diagnosis Related Groups (APR-DRG) to calculate SOI and ROM on hospitalized patients. APR-DRG data, including SOI and ROM, were gathered from individual chart reviews from July 6, 2020, to March 31, 2022.n = 103) and the 30-day mortality rate was 1.8% (n = 17). There were no in-program patient deaths. Out of 923 patients discharged from ACH, the average APR-DRG SOI was 2.89 (SD 0.81) and ROM was 2.73. (SD 0.92). Mean age was 70.88 (SD 14.46) years, 54.6% were male patients and the average length of stay was 4.10 days. The most frequent diagnosis was COVID-19 infection with 162 patients (17.6%), followed by heart failure exacerbation (12.7%) and septicemia (10.9%). The 30-day readmission rate after discharge from ACH was 11.2% (SOI and ROM from patients at the ACH program have been shown to be in the range of “moderate/major” according to the APR-DRG classification. The ACH program is capable of accepting and managing highly complex patients that require advanced therapeutic means. Furthermore, the ACH program has an in-program mortality rate of 0 to date. Therefore, ACH is rising as a capable alternative to the brick-and-mortar hospital.The online version contains supplementary material available at 10.1186/s12913-023-09333-7. Delivering high-acuity hospital level care at home has increased steadily over the last decade . This efThe All Patient Refined-Diagnosis Related Groups (APR-DRG) system is used to measure hospitalized patient disease burden and has two subclasses, Severity of Illness (SOI) and Risk of Mortality (ROM) that are used by many United States (US) hospital systems . APR-DRGAdvanced Care at Home (ACH) is the Mayo Clinic virtual hybrid HaH program which began treating patients in 2020. This program treats multiple Mayo Clinic patients across the US, with medical care planning directed by providers in a command center located in Jacksonville Florida and executed through in-home services delivered by local medical suppliers . This moThis study was approved by the Mayo Clinic Institutional Review Board as a retrospective chart review under protocol number 20–010753 and analyzed the de-identified patient data under protocol number 21–004666. The study was conducted between July 6, 2020, and March 31, 2022, at three Mayo Clinic Hospitals: Mayo Clinic in Florida, a 304-bed community academic hospital in Jacksonville, Florida, Mayo Clinic Healths Systems Eau Claire, a 304-bed community hospital in Eau Claire, Wisconsin, and Mayo Clinic in Arizona, a 268-bed community academic hospital in Phoenix, Arizona. The inclusion criteria for this study were the following: 1) all patients admitted to the ACH program in Florida, Wisconsin, and Arizona, with no age restrictions. Patients were excluded from the study if they did if they had missing or unknown data. Admission to the ACH program is completely voluntary. Patients provide both oral and written consent to participate in the ACH program and all subsequent research and experience studies.The ACH model of care has been described in detail previously , 12. BriPatients were monitored from the comfort of their homes using a technology stack and a specially configured audio/video communication device to communicate with their clinical teams. Patients received in home care from advanced practice provider visits, rapid response services, phlebotomy, nursing care, meals, and diagnostic images such as abdominal and chest radiographs as they would have received in the BAM hospital setting. All ACH patients are seen twice daily by either registered nurse or a community paramedic overseen by the command center virtual registered nurse. Laboratory studies and basic radiographic exams, such as x-rays and ultrasound, are performed in the home. Intravenous medications are administered by a visiting nurse or community paramedic. Respiratory and physical therapy services can be done in the home and the medical plan overseen and adjusted by the virtual care team. When the patients reached a stable endpoint, they were discharged from the program with regular primary care provider follow-up.We used APR-DRG to calculate the severity of illness (SOI) and risk of mortality (ROM) among patients enrolled in ACH. With the help of APR-DRG, metrics regarding SOI and ROM were calculated using EPIC Healthy Planet  and Optum (Optum One) to extract the data. Primary diagnostic related groups (DRG) were identified and divided into several categories such as: cardiology, gastroenterology, neurology, pulmonary, among others. Data collection was extracted from medical charts, administrative record abstractions, and insurance claims to document diagnoses. In addition, demographic characteristics including patient age, sex, and average length of stay (ALOS) were collected. Thirty-day readmission and mortality rates were collected; the 30-day period begins from the point of discharge from ACH. If a patient care escalation requiring return to the brick-and-mortar hospital occurred while the patient was enrolled in ACH, this did not count as a readmission as it was still part of the initial inpatient encounter. In-program mortality rate was also collected.p-value of < 0.05 being considered statistically significant.After data extraction from the electronic medical record regarding demographic characteristics and classification of each patient into one of the diagnostic related groups, an average and standard deviation (SD) measurements were performed of the independent variables of age, SOI and ROM using SPSS Statistics . The SOI and ROM of the COVID-19 and non-COVID-19 patients were calculated and compared using the unpaired Mann–Whitney U-test with a n = 103) and the 30-day mortality rate was 1.8% (n = 17). In addition, the average SOI was 2.89 (SD 0.81), and the ROM was 2.73 (SD 0.92) which stands in a range between “Moderate” and “Major” in the APR-DRG classification (Table n = 495), followed by cardiovascular disease (14.2%), surgical diagnosis (6.8%), airway diseases (5.5%), gastrointestinal / hepatobiliary disease (5.5%), kidney and urologic disease (5.2%), hematologic and oncologic disease (2.6%), musculoskeletal disease (2.1%), and endocrine disease (2.1%) , 54.3% were male, and the average LOS was 4.10 days. The 30-day readmission rate after discharge from ACH was 11.2% (n= 162), we decided to run a second analysis excluding these patients to determine if the SOI and ROM were inflated in value due to the number of COVID-19 patients, as it has been reported that hospitalized COVID-19 patients have higher ROM and SOI [n = 162) had a SOI of 3.41 and a ROM of 3.35, which was significantly higher than the non-COVID-19 cohort (p < 0.001)  was 3.41 and 3.35 respectively, supporting the belief that this cohort of patients is severely ill. Providers that did treat COVID-19 patients in HaH models may not have realized how severely ill they truly were, giving them more comfort to treat other high-acuity diagnosis at home. Second, we found that although the COVID-19 cohort had significantly higher SOI and ROM scores, even with this cohort of patients removed there was not a large decrease in our SOI or ROM (2.89 to 2.79 and 2.73 to 2.61 respectively), indicating that acuity of the non-COVID-19 patients still falls in the “moderate” to “major” category.Many providers may have only recently had an experience with HaH or advanced home telemedicine in a single diagnosis group (COVID-19) during the recent pandemic. Some may be more comfortable with the capabilities of treating COVID-19 patients in this capacity but possibly not other diagnosis. Others might question if our findings were inflated by the known high SOI and ROM associated with the COVID-19 APR-DRG. Several of our findings in our second data analysis may address these concerns. First, we found that the average SOI and ROM of our COVID-19 patients  or if the hospital consistently under or over reports risk factor(s) with influential effects on the model estimates and thus, alter the metric calculations. Lastly, if the statistical model is not properly calibrated and tested for systematic over or under estimation, then the model may systematically overestimate or underestimate expected values.The Advanced Care at Home virtual hybrid hospital-at-home program is capable of treating patients with “moderate” to “major” disease states, providing a realistic alternative for high-acuity care outside the brick-and-mortar hospital setting. Further investigations into patient acuity may both help build a more robust acceptance for ACH in patients with complex medical diseases as well as help standardize resource needs for patients in HaH programs with different acuity levels.Additional file 1: Appendix A. Clinical Stability Screening Tool.